animals Article Measurement of Calprotectin (S100A8/A9) in the Saliva of Pigs: Validation Data of A Commercially Available Automated Assay and Changes in Sepsis, Inflammation, and Stress María José López-Martínez 1 , Silvia Martínez-Subiela 1 , José Joaquín Cerón 1 , Alba Ortín-Bustillo 1 , Guillermo Ramis 2 , Marina López-Arjona 3 , Silvia Martínez-Miró 2 , Edgar García Manzanilla 4,5 , Peter David Eckersall 1,6 , Fernando Tecles 1 , Damián Escribano 1,2, * and Alberto Muñoz-Prieto 1 1 Interdisciplinary Laboratory of Clinical Analysis (INTERLAB-UMU), Department of Animal Medicine and Surgery, Veterinary School, Regional Campus of International Excellence Mare Nostrum, University of Murcia, 30100 Murcia, Spain; mariajose.lopez28@um.es (M.J.L.-M.); silviams@um.es (S.M.-S.); jjceron@um.es (J.J.C.); alba.ortinb@um.es (A.O.-B.); david.eckersall@glasgow.ac.uk (P.D.E.); ftecles@um.es (F.T.); alberto.munoz@um.es (A.M.-P.) 2 Department of Animal Production, Veterinary School, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, 30100 Murcia, Spain; guirramis@um.es (G.R.); silviamm@um.es (S.M.-M.) 3 Department of Animal and Food Science, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; marina.lopez.arjona@uab.cat 4 Pig Development Department, The Irish Food and Agriculture Authority, Teagasc, Moorepark, P61 C996 Fermoy, Ireland; egmanzanilla@gmail.com 5 School of Veterinary Medicine, University College Dublin, Belfield, D04 W6F6 Dublin, Ireland 6 School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Bearsden Rd., Glasgow G61 1QH, UK Citation: López-Martínez, M.J.; * Correspondence: det20165@um.es Martínez-Subiela, S.; Cerón, J.J.; Ortín-Bustillo, A.; Ramis, G.; Simple Summary: Calprotectin (CALP, S100A8/A9) is a calcium and zinc-binding protein involved López-Arjona, M.; Martínez-Miró, S.; in inflammation that has a wide range of proinflammatory functions, such as cytokine production Manzanilla, E.G.; Eckersall, P.D.; and regulation of leukocyte adhesion, migration, and phagocytosis. The objective of this study was to Tecles, F.; et al. Measurement of Calprotectin (S100A8/A9) in the validate a commercially available assay for the measurement of CALP in the saliva of pigs and study Saliva of Pigs: Validation Data of A the variations of this analyte due to sepsis, non-septic inflammation, and stress. The assay showed Commercially Available Automated adequate precision and accuracy for the measurements of CALP in the saliva of pigs. In addition, Assay and Changes in Sepsis, this protein showed significant increases in the saliva of pigs with sepsis as well as after a stressful Inflammation, and Stress. Animals situation in our experimental conditions, being the increase in the stress of lower magnitude than in 2023, 13, 1190. https://doi.org/ sepsis. Based on these results, CALP can be measured in the saliva of pigs and could be a potential 10.3390/ani13071190 biomarker of health and welfare in this species. Academic Editors: Isabel Henning-Pauka and Alexandra Abstract: Calprotectin (CALP, S100A8/A9), also named myeloid-related protein 8/14, is a dimer complex von Altrock of S100A8 and S100A9 that belongs to the S-100 protein family. It is involved in inflammation and has a wide range of proinflammatory functions, such as cytokine production and regulation of leukocyte Received: 10 March 2023 adhesion, migration, and phagocytosis. In humans, CALP traditionally can be measured in faeces, Revised: 24 March 2023 serum, and saliva as a biomarker of inflammation and sepsis. The objective of this study was to validate Accepted: 27 March 2023 an automated assay for CALP measurements in the saliva of pigs, having the advantage of the use of a Published: 29 March 2023 non-invasive sample that is easy to collect. The assay was precise and accurate. CALP in saliva measured by this assay showed significant changes depending on the hour of the day. It also showed significant increases in the saliva of pigs after the administration of lipopolysaccharide (LPS), and showed a rise, Copyright: © 2023 by the authors. although with increases of lower magnitude, after a stressful stimulus. Further studies should be made Licensee MDPI, Basel, Switzerland. to gain knowledge about the possible practical applications of the measurements of CALP in the saliva This article is an open access article of pigs as a biomarker to evaluate the animals’ health and welfare. distributed under the terms and conditions of the Creative Commons Keywords: S100A8/A9; calprotectin; automated assay; pig; saliva; stress; sepsis Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Animals 2023, 13, 1190. https://doi.org/10.3390/ani13071190 https://www.mdpi.com/journal/animals Animals 2023, 13, 1190 2 of 10 1. Introduction Calprotectin (CALP, S100A8/A9), also named myeloid-related protein 8/14, is a dimer complex of S100A8 and S100A9 which belongs to the S-100 protein family [1]. It is a calcium and zinc-binding protein involved in inflammation and has a wide range of proinflammatory functions, such as cytokine production and regulation of leukocyte adhesion, migration, and phagocytosis [2]. Currently, there is an increasing interest in the use of saliva as a biological sample since it has the advantage of being a non-invasive sample that is easy to collect. This is highly relevant in pigs, a species in which blood sampling is difficult, highly stressful, and painful for the animal. The collection of saliva can be easily made by farm personnel, allowing a more frequent analysis and monitoring to take place at the farm level. Currently, one of the main uses of saliva is for the diagnosis and detection of infectious diseases, but saliva can also be used to measure biomarkers that can provide information on stress, inflammation, immune response, and redox homeostasis [3]. Therefore, saliva contains a source of analytes with the potential to assess the effect of different husbandry conditions and also to evaluate the homeostasis in swine. In humans, CALP traditionally has been measured in faeces for the detection of inflammatory bowel disease and in serum as a biomarker of inflammation and sepsis [4]. However, it can also be quantified in saliva, where CALP has been reported to be increased in patients with active inflammatory bowel disease, suggesting that intestinal inflammation leads to increases in CALP in this sample type [5]. In pigs, CALP protein levels have been measured in faeces, increasing in animals with colitis [6–8]. In addition, variations in gene expression for calprotectin have also been found in the ileum after oral vaccination against E. coli [9]. However, to the authors’ knowledge, there are no reports about the measurement of CALP in saliva, despite the advantages that this sample type has. The objectives of this report were to perform an analytical validation of an automated commercially available assay for the measurement of CALP in the saliva of pigs and to determine if it is influenced by possible variations due to the sampling time during the day. In addition, the variations of this analyte due to sepsis, non-septic inflammation and stress were studied. For this purpose, the CALP concentration was measured in the saliva of pigs with three different conditions: sepsis experimentally induced by inoculation with lipopolysaccharide (LPS), non-septic inflammation induced by turpentine injection and a situation of stress such as staying at the lairage for 4 h after arrival at the slaughterhouse. In the models of LPS and turpentine, the correlation between values of CALP in saliva and CALP and C-reactive protein (CRP) in serum was studied. It is expected that this information will contribute to a better knowledge of the pathophysiology of this protein and its possible use as a biomarker of these conditions in saliva. 2. Materials and Methods 2.1. Assay Salivary CALP was determined by the BÜHLMANN fCal Turbo® assay (BÜHLMANN, Laboratories AG, Switzerland) which is an immunoturbidimetric assay using polystyrene nanoparticles coated with polyclonal anti-CALP antibodies. The assay was initially cal- ibrated with serial dilutions of a control material with a known concentration of CALP (Gentian, Moss, Norway) (Calibrator A). Then, a secondary calibrator (Calibrator B) con- sisting of a pool of saliva samples, in which the CALP concentration was determined with Calibrator A, was used for the complete validation and subsequent sample analysis. The CALP assay was validated for use in porcine saliva samples using aliquots of the saliva of pigs of the sepsis and non-septic inflammation experiment (Section 2.3). The validation of the assays was performed as follows based on previous procedures [10]: • Precision: the intra- and inter-assay coefficients of variation (CVs) were assessed using saliva samples with high and low CALP concentrations. • Accuracy: It was indirectly studied by evaluating linearity after serial dilutions with ultrapure water of saliva and samples with a high level of CALP. Additionally, recovery Animals 2023, 13, 1190 3 of 10 studies were made to see if there was a matrix effect in the determinations. For this, purified CALP (control material from Gentian, Moss, Norway) was used to spike saliva samples to reach three different CALP concentrations. • The lower limit of quantification (LLQ): it was calculated as the lowest CALP concen- tration that the assay was able to determine with an intra-assay CV < 20%. • Limit of detection (LD): based on the lowest concentration of CALP that the assays can distinguish from a specimen of zero value (ultrapure water), calculated as a mean value plus 3 standard deviations of 12 replicate determinations. 2.2. Evaluation of the Sampling in the Daytime To determine the effect of the time of sampling, surplus saliva samples from 20 male large white pigs aged 5 months from a commercial farm stored from a previous study were used [11]. In these pigs, saliva was sampled at 8 a.m., 12 a.m., 4 p.m., and 8 p.m. within the same day. In this experiment (and in all trials of the report), saliva was collected using Salivette tubes (Salivette, Sarstedt, Aktiengesellschaft & Co., Nümbrecht, Germany). Pigs chewed a sponge for 1 min. Each sponge was then placed in a Salivette tube and stored on ice until arrival at the laboratory. At the laboratory, the tubes were centrifuged at 3000× g for 10 min. Saliva samples were kept frozen at −80 ◦ C until analysis. 2.3. Experimental Sepsis and Non-Septic Inflammation Induction To evaluate the changes in CALP concentration caused by sepsis and non-sepsis inflammatory conditions, samples stored from a previous study were used [12]. A total of 15 growing male pigs, in the mid-fattening period, from the University of Murcia Farm, were included in this study. They had water ad libitum and a balanced diet and had a minimum space of 0.65 m2 per animal (Council Directive 2001/88/CE of 23 October 2001) with a mean temperature of 24 ± 2 ◦ C. The pigs were 14 weeks old and their median weight was 51.5 kg (interquartile range 48–53 kg). The pigs were adapted to the experimental conditions for a week and then randomized and divided into three groups of 5 animals each. A control group (n = 5) received saline treatment (2 mL) by an intramuscular route. An LPS group (n = 5) received a single dose of 30 µg/kg LPS from Escherichia Coli (LPS; O55:B5, Sigma-Aldrich, St. Louis, MO, USA) in sterile saline solution by intramuscular injection. A turpentine (TURP) group (n = 5) received 8 mL of TURP (oil of turpentine purified, Sigma–Aldrich) by two 4 mL subcutaneous injections in each front flank per animal. The injections were completed between 8 and 9 a.m. Saliva and blood samples were obtained 24 h before (baseline) the saline, LPS, or TURP injections and at 6, 24, and 48 h after the treatments. Basal, 24 h, and 48 h samples were obtained at 8 a.m. Saliva was collected and processed as indicated in Section 2.2. Blood samples were collected by venepuncture in an EDTA and plain tubes and serum were separated and stored at −80 ◦ C. 2.4. Stress Situation A total of 13 male pigs at the end-fattening period (5–6 months of age and mean body weight 105.2 ± 7.3 kg) were included in this study. They were from a farm in Southern Spain, where animals were housed in groups of 14 animals per pen (a minimum space of 0.65 m2 per animal) and given ad libitum access to a balanced diet and water. The pigs were transported to a commercial slaughterhouse at 15 km from the commercial farm. Transportation was undertaken during the spring of 2022, between 9 and 10 a.m., under commercially accepted conditions. The animals were unloaded on arrival at the slaughterhouse and placed in a lairage area (10 animals per pen) with free access to water. Saliva samples were collected at arrival at the slaughter on the day of transport (T0) (approximately at 10 a.m.) and 4 h after the transport (approximately at 2 p.m.) (T4). The transport of animals was according to the recommendations described in Directive 2001/88/EC, 2001 and Directive 2001/93/EC, 2001. Animals 2023, 13, 1190 4 of 10 Saliva samples were obtained as described in Section 2.2. Salivary cortisol concentra- tions of each animal were measured in these samples with a validated assay [13]. All the experimental procedures of this study were approved by the Ethical Committee on Animal Experimentation (CEEA) of the University of Murcia (A13220196; approval date: 4 March 2021) according to the European Council Directives considering the protection of animals used for experimental purposes. In addition, this study complies with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for the care and use of animals. 2.5. Statistical Analysis GraphPad Prism software Inc. (GraphPad Prism, version 8 for Windows, Graph Pad Software Inc., San Diego, CA, USA) was used to analyse the data. The D’Agostino and Pearson test was used to evaluate the data distribution giving a nonparametric distribution in all analyses. To analyse the effect of the day on salivary CALP concentrations and the effect of treatment in the LPS, TURP and saline (control) group, the Friedman test was performed, followed by Dunn’s multiple comparison tests to compare the groups over time. The comparison between the groups of pigs after transportation was performed by using the Wilcoxon matched pairs signed rank test. Correlations between salivary CALP and serum CALP and CRP (data of serum CRP in these animals have been reported in a previous study [12]) were analysed through the non-parametric Spearman correlation test. Moreover, in the stress situation, the correlation between salivary CALP and cortisol was evaluated by the same test. A correlation was strong when the correlation coefficient was ≥0.7. The results were considered significant if p-values were <0.05. 3. Results 3.1. Saliva Calprotectin Assay Validations The evaluation of precision showed mean intra- and inter-assay CVs of 3.50 and 4.79% for salivary CALP (Table 1). In the accuracy assessment, we observed recovery rates ranging from 110% to 116.7% for salivary CALP measurements (Table 2). Furthermore, the serial dilution of saliva samples with a high concentration of CALP showed linear regression equations with a coefficient of correlation close to 1 (Supplementary Material: Figure S1). The LLQ was set at 0.01 mg/L for salivary CALP, and the LD of the assay could not be calculated since all measurements with ultrapure water gave a value of zero. Table 1. Precision study of the salivary calprotectin assays. (SD, standard deviation; CV, coefficient of variation). Method Comparison Samples Mean (mg/L) SD (mg/L) CV (%) Saliva Intra-assay High 1.54 0.03 2.14 Low 0.55 0.02 4.86 Inter-assay High 1.67 0.04 4.26 Low 0.43 0.03 6.23 Table 2. Recovery study of the calprotectin assay in saliva. % Analyte Expected (mg/L) Observed (mg/L) Recovery (%) 100 0 0.72 0.72 100 75 25 0.66 0.60 110 50 50 0.54 0.48 112.5 25 75 0.42 0.36 116.7 0 100 0.24 0.24 100 3.2. Evaluation of the Sampling Time on the Day The CALP concentrations in saliva showed a tendency to decrease during the day, being these changes significantly at 12 p.m. (median = 0.07 mg/L, range = 0.03–0.30 mg/L) 50 50 0.54 0.48 112.5 25 75 0.42 0.36 116.7 0 100 0.24 0.24 100 Animals 2023, 13, 1190 3.2. Evaluation of the Sampling Time on the Day 5 of 10 The CALP concentrations in saliva showed a tendency to decrease during the day, being these changes significantly at 12 p.m. (median = 0.07 mg/L, range = 0.03–0.30 mg/L) and and88p.m. p.m.(median (median= 0.07 mg/L, = 0.07 range mg/L, = 0.03–0.72 range mg/L)mg/L) = 0.03–0.72 if compared with values if compared at 8 a.m. with values at (median = 0.21 mg/L, 8 a.m. (median = 0.21range mg/L,= 0.01–0.96 mg/L) (Figure range = 0.01–0.96 mg/L)1). (Figure 1). Resultsof Figure1.1.Results Figure ofsalivary salivary calprotectin calprotectin concentrations concentrations in in 20 20male malepigs pigsobtained obtainedatatdifferent differenthours hoursof the of day. the day. p-value ** ** < <0.01. p-value 0.01.Dots Dotsare arerepresenting median representing median values valuesand andwhiskers whiskersthe 95% the 95% CI.CI. 3.3.Experimental 3.3. ExperimentalSepsis Sepsisand andNon-Septic Non-SepticInflammation InflammationInduction Induction Insaliva, In saliva,the theCALP CALPlevels levelswere weresignificantly significantlyhigher higherin inpigs pigsatat24 24hhafter afterLPS LPSinjection injection (median = 1.26 mg/L, range = 0.54–1.32 mg/L) compared to basal (pre-treatment) levels (median = 1.26 mg/L, range = 0.54–1.32 mg/L) compared to basal (pre-treatment) levels (median = 0.18, range = 0.06–0.24 mg/L) (p = 0.005), being reduced to nearly basal levels (median = 0.18, range = 0.06–0.24 mg/L) (p = 0.005), being reduced to nearly basal levels at at 48 h (Figure 2). In the case of the administration of TURP, a tendency of increase 6was Animals 2023, 13, x FOR PEER REVIEW of 11 48 h (Figure 2). In the case of the administration of TURP, a tendency of increase was observed after the inoculation, but the changes were not significant. The effect of saline observed after the inoculation, but the changes were not significant. The effect of saline injection did not cause any variation in salivary calprotectin levels. injection did not cause any variation in salivary calprotectin levels. Figure Figure 2. 2. Changes Changes inin the the salivary salivary calprotectin calprotectin concentrations concentrations ofof pigs pigs after after lipopolysaccharide lipopolysaccharide (LPS), (LPS), turpentine(TURP) turpentine (TURP)ororsaline saline(control) (control)injection. injection. The The plots plotsshow showmedians medians(line (linewithin within box), 25th box), and 25th 75th percentiles (boxes) and min and max values (whiskers). Asterisks indicate a statistically and 75th percentiles (boxes) and min and max values (whiskers). Asterisks indicate a statistically signif- icant difference: ** p < 0.01. significant difference: ** p < 0.01. InInserum, serum,pigs pigssubjected subjected to to LPS LPS injection injectionshowed showeda asignificant increase significant in CALP increase in CALPcon- centrations atat6 6h h(median concentrations (median= =0.17 0.17mg/L, mg/L,range = 0.09–0.36 range = 0.09–0.36mg/L) compared mg/L) comparedwith basal with val- basal ues (median = 0.02 mg/L, range = 0.01–0.03 mg/L) (p = 0.01). In the TURP group, values (median = 0.02 mg/L, range = 0.01–0.03 mg/L) (p = 0.01). In the TURP group, there there were no significant changes in the pairwise comparison although a tendency of increase at 6 h and 24 h compared with the control group was observed (Figure 3). 75th percentiles (boxes) and min and max values (whiskers). Asterisks indicate a statistically sign icant difference: ** p < 0.01. In serum, pigs subjected to LPS injection showed a significant increase in CALP co Animals 2023, 13, 1190 centrations at 6 h (median = 0.17 mg/L, range = 0.09–0.36 mg/L) compared with 6 of 10basal va ues (median = 0.02 mg/L, range = 0.01–0.03 mg/L) (p = 0.01). In the TURP group, there we no significant changes in the pairwise comparison although a tendency of increase at 6 were no significant changes in the pairwise comparison although a tendency of increase at and 24 h compared with the control group was observed (Figure 3). 6 h and 24 h compared with the control group was observed (Figure 3). Changes in Figure3.3.Changes Figure in the theserum serum calprotectin calprotectinconcentrations of pigsofafter concentrations pigslipopolysaccharide (LPS), after lipopolysaccharide (LP turpentine (TURP) or saline (control) injection. The plots show medians (line within turpentine (TURP) or saline (control) injection. The plots show medians (line within box), box), 25th and 25th an 75th 75th percentiles (boxes) percentiles (boxes) and andmin and min andmax values max (whiskers). values * p-value (whiskers). < 0.05. < 0.05. *p-value The correlation study showed that salivary and serum CALP concentrations were The correlation positively study correlated (r showed = 0.283, thatInsalivary p = 0.02). addition,and serum serum CALP concentrations CRP concentration values we positively correlated were significantly (r = 0.283, positively p = 0.02). correlated withInboth addition, serum salivary CRPpconcentration (r = 0.665, values we = 0.01) and serum significantly positively CALP concentrations correlated (r = with both 0.418, p < 0.001). salivary All these (r = 0.665, correlations werepbelow = 0.01) 0.7and serum which is CAL considered the (r concentrations threshold = 0.418,for p